SUMMARY The goal of this proposal is to define novel approaches to promote muscle stem cells (MuSC) expansion. MuSC are the major source of skeletal muscle growth and regeneration. This cell population is capable of self-renewal, i.e. generating large number of progenitors to repair damaged tissue and at the same time more copies of themselves upon cell division to maintain the stem cell pool. MuSC exist in healthy adult tissues in a quiescent state, and upon stress or injury they are activated to proliferate and repair the damaged tissue. While this process is efficient in healthy conditions, in several diseased states and during aging the function of MuSC is impaired and this significantly contribute to impaired tissue maintenance and function. However, their clinical utility as therapeutic tools and targets is currently hindered by our limited understanding of the molecular networks regulating their function. Thus, there is a major need to fill this gap and define novel approaches that can promote MuSC expansion and tissue repair, as these would find applications for disease modeling in vitro as well as for promoting MuSC-mediated tissue regeneration in chronic conditions. Our preliminary results provide evidence that the E3 ubiquitin ligase Fbxw7, which regulates multiple adult stem cell compartments, promotes MuSC commitment and differentiation, and its genetic ablation leads to robust MuSC expansion in vivo. The focus of the proposed studies is to dynamically map Fbxw7 protein targets during MuSC fate transitions, and to evaluate its transient inhibition as a novel approach to promote MuSC expansion. Our research will take advantage of (1) the inducible Pax7-CreER;Fbxw7f/f novel animal model, (2) an optimized global proteomics approach for MuSC profiling, (3) the use of Fbxw7 inhibitors for its transient manipulation, and (4) Human MuSC isolated from patients, to validate conservation across species. Together, this exploratory study will provide an initial understanding of how Fbxw7 impacts MuSC cell fate decisions and identify novel tools that can be utilized for regenerative medicine approaches to enhance MuSC expansion and tissue repair in diseased conditions.